Knobs and holes heteromeric polypeptides

ABSTRACT

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins, heteromultimers and antibody-immunoadhesin chimeras. Generally, the method involves introducing a protuberance at the interface of a first polypeptide and a corresponding cavity in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heteromultimer formation and hinder homomultimer formation. The protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/700,618 filed Feb. 4, 2010 (now U.S. Pat. No. 8,216,805 issued Jul.10, 2012), which is a continuation of U.S. application Ser. No.11/533,709 filed Sep. 20, 2006 (now U.S. Pat. No. 7,695,936 issued Apr.13, 2010), which is a continuation of U.S. application Ser. No.10/010,245 filed Dec. 7, 2001 (now U.S. Pat. No. 7,642,228 issue Jan. 5,2010), which is a continuation of application Ser. No. 08/974,183 filedNov. 19, 1997, now abandoned, which is a continuation of U.S.application Ser. No. 08/399,106 filed Mar. 1, 1995 (now U.S. Pat. No.5,731,168 issued Mar. 24, 1998), all of which are incorporated herein byreference and to which priority is claimed under 35 USC §120.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for making heteromultimericpolypeptides such as multispecific antibodies (e.g. bispecificantibodies), multispecific immunoadhesins (e.g. bispecificimmunoadhesins) as well as antibody-immunoadhesin chimeras and theheteromultimeric polypeptides made using the method.

2. Description of Related Art

Bispecific Antibodies

Bispecific antibodies (BsAbs) which have binding specificities for atleast two different antigens have significant potential in a wide rangeof clinical applications as targeting agents for in vitro and in vivoimmunodiagnosis and therapy, and for diagnostic immunoassays.

In the diagnostic areas, bispecific antibodies have been very useful inprobing the functional properties of cell surface molecules and indefining the ability of the different Fc receptors to mediatecytotoxicity (Fanger et al., Crit. Rev. Immunol. 12:101-124 [1992]).Nolan et al., Biochem. Biophys. Acta. 1040:1-11 (1990) describe otherdiagnostic applications for BsAbs. In particular, BsAbs can beconstructed to immobilize enzymes for use in enzyme immunoassays. Toachieve this, one arm of the BsAb can be designed to bind to a specificepitope on the enzyme so that binding does not cause enzyme inhibition,the other arm of the BsAb binds to the immobilizing matrix ensuring ahigh enzyme density at the desired site. Examples of such diagnosticBsAbs include the rabbit anti-IgG/anti-ferritin BsAb described byHammerling et al., J. Exp. Med. 128:1461-1473 (1968) which was used tolocate surface antigens. BsAbs having binding specificities for horseradish peroxidase (HRP) as well as a hormone have also been developed.Another potential immunochemical application for BsAbs involves theiruse in two-site immunoassays. For example, two BsAbs are producedbinding to two separate epitopes on the analyte protein—one BsAb bindsthe complex to an insoluble matrix, the other binds an indicator enzyme(see Nolan et al., supra).

Bispecific antibodies can also be used for in vitro or in vivoimmunodiagnosis of various diseases such as cancer (Songsivilai et al.,Clin. Exp. Immunol. 79:315 [1990]). To facilitate this diagnostic use ofthe BsAb, one arm of the BsAb can bind a tumor associated antigen andthe other arm can bind a detectable marker such as a chelator whichtightly binds a radionuclide. Using this approach, Le Doussal et al.made a BsAb useful for radioimmunodetection of colorectal and thyroidcarcinomas which had one arm which bound a carcinoembryonic antigen(CEA) and another arm which bound diethylenetriaminepentacetic acid(DPTA). See Le Doussal et al., Int. J. Cancer Suppl. 7:58-62 (1992) andLe Doussal et al., J. Nucl. Med. 34:1662-1671 (1993). Stickney et al.similarly describe a strategy for detecting colorectal cancersexpressing CEA using radioimmunodetection. These investigators describea BsAb which binds CEA as well as hydroxyethylthiourea-benzyl-EDTA(EOTUBE). See Stickney et al., Cancer Res. 51:6650-6655 (1991).

Bispecific antibodies can also be used for human therapy in redirectedcytotoxicity by providing one arm which binds a target (e.g. pathogen ortumor cell) and another arm which binds a cytotoxic trigger molecule,such as the T-cell receptor or the Fcÿ receptor. Accordingly, bispecificantibodies can be used to direct a patient's cellular immune defensemechanisms specifically to the tumor cell or infectious agent. Usingthis strategy, it has been demonstrated that bispecific antibodies whichbind to the Fcÿ RIII (i.e. CD16) can mediate tumor cell killing bynatural killer (NK) cell/large granular lymphocyte (LGL) cells in vitroand are effective in preventing tumor growth in vivo. Segal et al.,Chem. Immunol. 47:179 (1989) and Segal et al., Biologic Therapy ofCancer 2(4) DeVita et al. eds. J. B. Lippincott, Philadelphia (1992)p. 1. Similarly, a bispecific antibody having one arm which bindsFcÿRIII and another which binds to the HER2 receptor has been developedfor therapy of ovarian and breast tumors that overexpress the HER2antigen. (Hseih-Ma et al. Cancer Research 52:6832-6839 [1992] and Weineret al. Cancer Research 53:94-100 [1993]). Bispecific antibodies can alsomediate killing by T cells. Normally, the bispecific antibodies link theCD3 complex on T cells to a tumor-associated antigen. A fully humanizedF(ab′)₂ BsAb consisting of anti-CD3 linked to anti-p185^(HER2) has beenused to target T cells to kill tumor cells overexpressing the HER2receptor. Shalaby et al., J. Exp. Med. 175(4217 (1992). Bispecificantibodies have been tested in several early phase clinical trials withencouraging results. In one trial, 12 patients with lung, ovarian orbreast cancer were treated with infusions of activated T-lymphocytestargeted with an anti-CD3/anti-tumor (MOC31) bispecific antibody. deLeijet al. Bispecific Antibodies and Targeted Cellular Cytotoxicity,Romet-Lemonne, Fanger and Segal Eds., Lienhart (1991) p. 249. Thetargeted cells induced considerable local lysis of tumor cells, a mildinflammatory reaction, but no toxic side effects or anti-mouse antibodyresponses. In a very preliminary trial of an anti-CD3/anti-CD19bispecific antibody in a patient with B-cell malignancy, significantreduction in peripheral tumor cell counts was also achieved. Clark etal. Bispecific Antibodies and Targeted Cellular Cytotoxicity,Romet-Lemonne, Fanger and Segal Eds., Lienhart (1991) p. 243. See alsoKroesen et al., Cancer Immunol. Immunother. 37:400-407 (1993), Kroesenet al., Br. J. Cancer 70:652-661 (1994) and Weiner et al., J. Immunol.152:2385 (1994) concerning therapeutic applications for BsAbs.

Bispecific antibodies may also be used as fibrinolytic agents or vaccineadjuvants. Furthermore, these antibodies may be used in the treatment ofinfectious diseases (e.g. for targeting of effector cells to virallyinfected cells such as HIV or influenza virus or protozoa such asToxoplasma gondii), used to deliver immunotoxins to tumor cells, ortarget immune complexes to cell surface receptors (see Fanger et al.,supra).

Use of BsAbs has been effectively stymied by the difficulty of obtainingBsAbs in sufficient quantity and purity. Traditionally, bispecificantibodies were made using hybrid-hybridoma technology (Millstein andCuello, Nature 305:537-539 [1983]). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure (see FIG. 1 herein). Thepurification of the correct molecule, which is usually done by affinitychromatography steps, is rather cumbersome, and the product yields arelow. Accordingly, techniques for the production of greater yields ofBsAb have been developed. These are depicted in FIGS. 2A-2E herein. Asshown in FIG. 2A, bispecific antibodies can be prepared using chemicallinkage. To achieve chemical coupling of antibody fragments, Brennan etal., Science 229:81 (1985) describe a procedure wherein intactantibodies are proteolytically cleaved to generate F(ab′)₂ fragments.These fragments are reduced in the presence of the dithiol complexingagent sodium arsenite to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the BsAb. The BsAbs produced can beused as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli. which can be chemically coupled to form bispecificantibodies (see FIG. 2B). Shalaby et al., J. Exp. Med. 175:217-225(1992) describe the production of a fully humanized BsAb F(ab′)₂molecule having one arm which binds p185^(HER2) and another arm whichbinds CD3. Each Fab′ fragment was separately secreted from E. coli. andsubjected to directed chemical coupling in vitro to form the BsAb. TheBsAb thus formed was able to bind to cells overexpressing the HER2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets. Seealso Rodrigues et al., Int. J. Cancers (Suppl.) 7:45-50 (1992).

Various techniques for making and isolating BsAb fragments directly fromrecombinant cell cultures have also been described. For example,bispecific F(ab′)₂ heterodimers have been produced using leucine zippers(see FIG. 2C). Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of anti-CD3 and anti-interleukin-2 receptor (IL-2R)antibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then reoxidized to form the antibodyheterodimers. The BsAbs were found to be highly effective in recruitingcytotoxic T cells to lyse HuT-102 cells in vitro. The advent of the“diabody” technology described by Hollinger et al., PNAS (USA)90:6444-6448 (1993) has provided an alternative mechanism for makingBsAb fragments. The fragments comprise a heavy chain variable domain(V_(H)) connected to a light chain variable domain (V_(L)) by a linkerwhich is too short to allow pairing between the two domains on the samechain. Accordingly, the V_(H) and V_(L) domains of one fragment areforced to pair with the complementary V_(L) and V_(H) domains of anotherfragment, thereby forming two antigen-binding sites (see FIG. 2Dherein). Another strategy for making BsAb fragments by the use of singlechain Fv (sFv) dimers has also been reported. See Gruber et al. J.Immunol. 152: 5368 (1994). These researchers designed an antibody whichcomprised the V_(H) and V_(L) domains of an antibody directed againstthe T cell receptor joined by a 25 amino acid residue linker to theV_(H) and V_(L) domains of an anti-fluorescein antibody. The refoldedmolecule (see FIG. 2E herein) bound to fluorescein and the T cellreceptor and redirected the lysis of human tumor cells that hadfluorescein covalently linked to their surface.

It is apparent that several techniques for making bispecific antibodyfragments which can be recovered directly from recombinant cell culturehave been reported. However, full length BsAbs may be preferable to BsAbfragments for many clinical applications because of their likely longerserum half-life and possible effector functions.

Immunoadhesins

Immunoadhesins (Ia's) are antibody-like molecules which combine thebinding domain of a protein such as a cell-surface receptor or a ligand(an “adhesin”) with the effector functions of an immunoglobulin constantdomain. Immunoadhesins can possess many of the valuable chemical andbiological properties of human antibodies. Since immunoadhesins can beconstructed from a human protein sequence with a desired specificitylinked to an appropriate human immunoglobulin hinge and constant domain(Fc) sequence, the binding specificity of interest can be achieved usingentirely human components. Such immunoadhesins are minimally immunogenicto the patient, and are safe for chronic or repeated use.

Immunoadhesins reported in the literature include fusions of the T cellreceptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940[1987]); CD4 (Capon et al., Nature 337:525-531 [1989]; Traunecker etal., Nature 339:68-70 [1989]; Zettmeissl et al., DNA Cell Biol. USA9:347-353 [1990]; and Byrn et al., Nature 344:667-670 [1990]);L-selectin or homing receptor (Watson et al., J. Cell. Biol.110:2221-2229 [1990]; and Watson et al., Nature 349:164-167 [1991]);CD44 (Aruffo et al., Cell 61:1303-1313 [1990]); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 [1991]); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 [1991]); CD22 (Stamenkovic et al., Cell 66:1133-1144[1991]); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 [1991]; Lesslauer et al., Eur. J. Immunol. 27:2883-2886[1991]; and Peppel et al., J. Exp. Med. 174:1483-1489 [1991]); NPreceptors (Bennett et al., J. Biol. Chem. 266:23060-23067 [1991]);inteferon ÿ receptor (Kurschner et al., J. Biol. Chem. 267:9354-9360[1992]); 4-1BB (Chalupny et al., PNAS [USA] 89:10360-10364 [1992]) andIgE receptor ÿ (Ridgway and Gorman, J. Cell. Biol. Vol. 115, AbstractNo. 1448 [1991]).

Examples of immunoadhesins which have been described for therapeutic useinclude the CD4-IgG immunoadhesin for blocking the binding of HIV tocell-surface CD4. Data obtained from Phase I clinical trials in whichCD4-IgG was administered to pregnant women just before delivery suggeststhat this immunoadhesin may be useful in the prevention ofmaternal-fetal transfer of HIV. Ashkenazi et al., Intern. Rev. Immunol.10:219-227 (1993). An immunoadhesin which binds tumor necrosis factor(TNF) has also been developed. TNF is a proinflammatory cytokine whichhas been shown to be a major mediator of septic shock. Based on a mousemodel of septic shock, a TNF receptor immunoadhesin has shown promise asa candidate for clinical use in treating septic shock (Ashkenazi et al.,supra). Immunoadhesins also have non-therapeutic uses. For example, theL-selectin receptor immunoadhesin was used as an reagent forhistochemical staining of peripheral lymph node high endothelial venules(HEV). This reagent was also used to isolate and characterize theL-selectin ligand (Ashkenazi et al., supra).

If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., J. Immunol. Methods162:123 (1993) describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin. Binding studies indicated that the bispecific immunoglobulinfusion protein so formed had an enhanced ability to bind to a myeloidcell line compared to the monospecific immunoadhesins from which it wasderived.

Antibody-Immunoadhesin Chimeras

Antibody-immunoadhesin (Ab/Ia) chimeras have also been described in theliterature. These molecules combine the binding region of animmunoadhesin with the binding domain of an antibody.

Berg et al., PNAS (USA) 88:4723-4727 (1991) made a bispecificantibody-immunoadhesin chimera which was derived from murine CD4-IgG.These workers constructed a tetrameric molecule having two arms. One armwas composed of CD4 fused with an antibody heavy-chain constant domainalong with a CD4 fusion with an antibody light-chain constant domain.The other arm was composed of a complete heavy-chain of an anti-CD3antibody along with a complete light-chain of the same antibody. Byvirtue of the CD4-IgG this bispecific molecule binds to CD3 on thesurface of cytotoxic T cells. The juxtaposition of the cytotoxic cellsand HIV-infected cells results in specific killing of the latter cells.

While Berg et al. describe a bispecific molecule that was tetrameric instructure, it is possible to produce a trimeric hybrid molecule thatcontains only one CD4-IgG fusion. See Chamow et al., J. Immunol.153:4268 (1994). The first arm of this construct is formed by ahumanized anti-CD3 ÿ light chain and a humanized anti-CD3 ÿ heavy chain.The second arm is a CD4-IgG immunoadhesin which combines part of theextracellular domain of CD4 responsible for gp120 binding with the Fcdomain of IgG. The resultant Ab/Ia chimera mediated killing ofHIV-infected cells using either pure cytotoxic T cell preparations orwhole peripheral blood lymphocyte (PBL) fractions that additionallyincluded Fc receptor-bearing large granular lymphocyte effector cells.

In the manufacture of the above-mentioned heteromultimers, it isdesirable to increase the yields of the desired heteromultimer over thehomomultimer(s). The invention described herein provides a means forachieving this.

SUMMARY OF THE INVENTION

This application describes a “protuberance-into-cavity” strategy whichserves to engineer an interface between a first and second polypeptidefor hetero-oligomerization. See FIG. 4 for a schematic illustration ofthe strategy employed. The preferred interface comprises at least a partof the C_(H)3 domain of an antibody constant domain. “Protuberances” areconstructed by replacing small amino acid side chains from the interfaceof the first polypeptide with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to theprotuberances are optionally created on the interface of the secondpolypeptide by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). Where a suitably positioned and dimensionedprotuberance or cavity exists at the interface of either the first orsecond polypeptide, it is only necessary to engineer a correspondingcavity or protuberance, respectively, at the adjacent interface.

Accordingly, the invention can be said to relate to a method ofpreparing a heteromultimer comprising a first polypeptide and a secondpolypeptide which meet at an interface, wherein the first polypeptidehas a protuberance at the interface thereof which is positionable in acavity at the interface of the second polypeptide. In one aspect, themethod involves: (a) culturing a host cell comprising nucleic acidencoding the first polypeptide and second polypeptide, wherein thenucleic acid encoding the first polypeptide has been altered from theoriginal nucleic acid to encode the protuberance or the nucleic acidencoding the second polypeptide has been altered from the originalnucleic acid to encode the cavity, or both, such that the nucleic acidis expressed; and (b) recovering the heteromultimer from the host cellculture.

Normally, the nucleic acid encoding both the first polypeptide and thesecond polypeptide are altered to encode the protuberance and cavity,respectively. Preferably the first and second polypeptides each comprisean antibody constant domain such as the C_(H)3 domain of a human IgG₁.

The invention also provides a heteromultimer (such as a bispecificantibody, bispecific immunoadhesin or antibody/immunoadhesin chimera)comprising a first polypeptide and a second polypeptide which meet at aninterface. The interface of the first polypeptide comprises aprotuberance which is positionable in a cavity in the interface of thesecond polypeptide, and the protuberance or cavity, or both, have beenintroduced into the interface of the first and second polypeptidesrespectively. The heteromultimer may be provided in the form of acomposition further comprising a pharmaceutically acceptable carrier.

The invention also relates to a host cell comprising nucleic acidencoding the heteromultimer of the preceding paragraph wherein thenucleic acid encoding the first polypeptide and second polypeptide ispresent in a single vector or in separate vectors. The host cell can beused in a method of making a heteromultimer which involves culturing thehost cell so that the nucleic acid is expressed and recovering theheteromultimer from the cell culture.

In yet a further aspect, the invention provides a method of preparing aheteromultimer comprising:

(a) altering a first nucleic acid encoding a first polypeptide so thatan amino acid residue in the interface of the first polypeptide isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance on the first polypeptide;

(b) altering a second nucleic acid encoding a second polypeptide so thatan amino acid residue in the interface of the second polypeptide isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity in the second polypeptide, wherein theprotuberance is positionable in the cavity;

(c) introducing into a host cell the first and second nucleic acids andculturing the host cell so that expression of the first and secondnucleic acid occurs; and

(d) recovering the heteromultimer formed from the cell culture.

The invention provides a mechanism for increasing the yields of theheteromultimer over other unwanted end-products such as homomultimers.Preferably, the yields of the heteromultimer recovered from recombinantcell culture are at least greater than 80% and preferably greater than90% compared to the by-product homomultimer(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the various antibody molecules which may be generatedwhen the traditional hybrid-hybridoma technique of Millstein and Cuello,supra, is used for making full length BsAbs.

FIGS. 2A-2E illustrate the various techniques of the background art formanufacturing BsAb fragments, reviewed in the background section above.

FIGS. 3A-3C depict an exemplary strategy for making an immunoadhesindimer (SEQ ID NO.:1) (FIG. 3C) comprising the binding domain of areceptor (FIG. 3A) and the constant domain of an IgG₁ immunoglobulin(FIG. 3B).

FIG. 4 illustrates schematically the protuberance-into-cavity strategyof the instant application for generating heteromultimers.

FIG. 5 shows the interface residues of the C_(H)3 domain of theimmunoglobulins IgG (SEQ ID NO.:2), IgA (SEQ ID NO.:3), IgD (SEQ IDNO.:4), IgE (SEQ ID NO.:5) and IgM (SEQ ID NO.:6). The C_(H)3 domain ofeach of these immunoglobulins is made up of a “ÿ-sandwich”, which iscomprised of two separate and parallel “ÿ-sheets”. One of the ÿ-sheetsprovides the interface residues, the other is the “exterior ÿ-sheet”.The ÿ-sheet forming the interface is formed from four “ÿ-strands”. Theresidues of each of the seven ÿ-strands of the C_(H)3 domain of thevarious immunoglobulins are identified by dashed overlining. Theresidues in the middle and edge ÿ-strands of the interface areidentified, as are those of the exterior ÿ-sheet. Residue numbering isaccording to Fc crystal structure (Deisenhofer, Biochem. 20:2361[1981]). The residues buried in the interior of the C_(H)3 domain areidentified with a “B”, those which are partially buried in the interiorof the C_(H)3 domain are identified with a “b”, those “contact” residueswhich are partially buried at the interface (i.e. 26%-10% exposed) areidentified with an “i” and those which are buried at the interface (i.e.<6% exposed) are identified with an “I”. The bold residues are optimalcandidate original residues for replacement with import residues.

FIG. 6 identifies the interface residues of human (h) (‘hIgG1’, ‘hIgG2’,‘hIgG3’, ‘hIgG4’, and ‘hIgE’ disclosed as SEQ ID NOs.:7-10 and 15,respectively, in order of appearance) or murine (m) (‘mIgG1’, ‘mIgG2A’,‘mIgG2B’ and ‘mIgG3’, disclosed as SEQ ID NOs.:11-14, respectively, inorder of appearance) IgG subtypes (B=ASX and Z=GLX). The residues inÿ-strands at the edge and middle of the interface are bracketed and“contact” residues are indicated with arrows. Sequences obtained fromMiller et al., J. Mol. Biol. 216:965 (1990) and Kabat et al., Sequencesof Proteins of Immunological Interest, National Institutes of Health,Bethesda, Md., ed. 5, (1991). It is apparent that the contact residuesare highly conserved.

FIG. 7 shows the interface residues of the C_(H)3 domain of human IgG₁SEQ ID NOs.:16-19, respectively, in order of appearance). Data derivedfrom Miller et al., J. Mol. Biol. 216:965 (1990). “Contact” residues areshown and those residues mutated in the examples described herein areboxed.

FIG. 8 shows schematically the co-transfection assay for examining Fcheterodimerization described in the examples.

FIG. 9 depicts a C_(H)3 dimer based upon a 2.9 Å structure of human IgG₁Fc (Deisenhofer, Biochem. 20:2361 [1981]) highlighting T366Y and Y407Tmutations on opposite sides of the interface together with residuesPhe⁴⁰⁵ and Thr³⁹⁴ (“Kabat numbering”—Kabat et al., Sequences of Proteinsof Immunological Interest, National Institutes of Health, Bethesda, Md.,ed. 5, [1991]).

FIGS. 10A-10E depict a scanning densitometric analysis of SDS-PAGE ofproducts from co-transfection of antibody (Ab) heavy (H) and light (L)chains with immunoadhesin (Ia). FIG. 10A shows wild-type; FIG. 10B showsmutant Ab Y407T, Ia T366Y; FIG. 10C shows mutant Ab T366Y, Ia Y407T;FIG. 10D shows mutant Ab F405A, Ia T394W; and FIG. 10E shows mutant AbT366Y:F405A, Ia T394W:Y407T. Data presented are the mean from at least 2independent experiments. The densitometric signal response was found tobe linear (R=0.9993) over the experimental range used (0.02-10 ÿg) asjudged by control experiment using a closely related humanized antibody,huMAb4D5-8 (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 [1992]).

I. DEFINITIONS

In general, the following words or phrases have the indicateddefinitions when used in the description, examples, and claims:

A “heteromultimer” or “heteromultimeric polypeptide” is a moleculecomprising at least a first polypeptide and a second polypeptide,wherein the second polypeptide differs in amino acid sequence from thefirst polypeptide by at least one amino acid residue. Preferably, theheteromultimer has binding specificity for at least two differentligands or binding sites. The heteromultimer can comprise a“heterodimer” formed by the first and second polypeptide or can formhigher order tertiary structures where polypeptides in addition to thefirst and second polypeptide are present. Exemplary structures for theheteromultimer include heterodimers (e.g. the bispecific immunoadhesindescribed by Dietsch et al., supra), heterotrimers (e.g. the Ab/Iachimera described by Chamow et al., supra), heterotetramers (e.g. abispecific antibody) and further oligomeric structures.

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. Preferably, mammalianpolypeptides (polypeptides that were originally derived from a mammalianorganism) are used, more preferably those which are directly secretedinto the medium. Examples of bacterial polypeptides include, e.g.,alkaline phosphatase and ÿ-lactamase. Examples of mammalian polypeptidesinclude molecules such as renin, a growth hormone, including humangrowth hormone; bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors;integrin; protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-ÿ;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-ÿ1, TGF-ÿ2, TGF-ÿ3,TGF-ÿ4, or TGF-ÿ5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides.

The “first polypeptide” is any polypeptide which is to be associatedwith a second polypeptide. The first and second polypeptide meet at an“interface” (defined below). In addition to the interface, the firstpolypeptide may comprise one or more additional domains, such as“binding domains” (e.g. an antibody variable domain, receptor bindingdomain, ligand binding domain or enzymatic domain) or antibody constantdomains (or parts thereof) including C_(H)2, C_(H)1 and C_(L) domains.Normally, the first polypeptide will comprise at least one domain whichis derived from an antibody. This domain conveniently is a constantdomain, such as the C_(H)3 domain of an antibody and can form theinterface of the first polypeptide. Exemplary first polypeptides includeantibody heavy chain polypeptides, chimeras combining an antibodyconstant domain with a binding domain of a heterologous polypeptide(i.e. an immunoadhesin, see definition below), receptor polypeptides(especially those which form dimers with another receptor polypeptide,e.g., interleukin-8 receptor [IL-8R] and integrin heterodimers [e.g.LFA-1 or GPIIIb/IIIa]), ligand polypeptides (e.g. nerve growth factor[NGF], neurotrophin-3 [NT-3], and brain-derived neurotrophic factor[BDNF]—see Arakawa et al. J. Biol. Chem. 269(45): 27833-27839 [1994] andRadziejewski et al. Biochem. 32(48): 1350 [1993]) and antibody variabledomain polypeptides (e.g. diabodies). The preferred first polypeptide isselected from an antibody heavy chain and an immunoadhesin.

The “second polypeptide” is any polypeptide which is to be associatedwith the first polypeptide via an “interface”. In addition to theinterface, the second polypeptide may comprise additional domains suchas a “binding domain” (e.g. an antibody variable domain, receptorbinding domain, ligand binding domain or enzymatic domain), or antibodyconstant domains (or parts thereof) including C_(H)2, C_(H)1 and C_(L)domains. Normally, the second polypeptide will comprise at least onedomain which is derived from an antibody. This domain conveniently is aconstant region, such as the C_(H)3 domain of an antibody and can formthe interface of the second polypeptide. Exemplary second polypeptidesinclude antibody heavy chain polypeptides, chimeras combining anantibody constant domain with a binding domain of a heterologouspolypeptide (i.e. an immunoadhesin, see definition below), receptorpolypeptides (especially those which form dimers with another receptorpolypeptide, e.g., interleukin-8 receptor [IL-8R] and integrinheterodimers [e.g. LFA-1 or GPIIIb/IIIa]), ligand polypeptides (e.g.nerve growth factor [NGF], neurotrophin-3 [NT-3], and brain-derivedneurotrophic factor [BDNF]—see Arakawa et al. J. Biol. Chem. 269(45):27833-27839 [1994] and Radziejewski et al. Biochem. 32(48): 1350 [1993])and antibody variable domain polypeptides (e.g. diabodies). Thepreferred second polypeptide is selected from an antibody heavy chainand an immunoadhesin.

A “binding domain” comprises any region of a polypeptide which isresponsible for selectively binding to a molecule of interest (e.g. anantigen, ligand, receptor, substrate or inhibitor). Exemplary bindingdomains include an antibody variable domain, receptor binding domain,ligand binding domain and an enzymatic domain.

The term “antibody” shall mean a polypeptide containing one or moredomains capable of binding an epitope on an antigen of interest, wheresuch domain(s) are derived from or homologous with the variable regionof an antibody. Examples of antibodies include full length antibodies,antibody fragments, single chain molecules, bispecific or bifunctionalmolecules, diabodies, and chimeric antibodies (e.g. humanized andPrimatized™ antibodies). “Antibody fragments” include Fv, Fv′, Fab,Fab′, and F(ab′)₂ fragments.

“Humanized” forms of non-human (e.g. rodent or primate) antibodies arespecific chimeric immunoglobulins, immunoglobulin chains or fragmentsthereof which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, rabbit or primate having the desired specificity, affinityand capacity. In some instances, Fv framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, the humanized antibody may comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. The humanized antibody includes a Primatized™antibody wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by immunizing macaque monkeys with the antigenof interest.

A “multispecific antibody” is a molecule having binding specificitiesfor at least two different antigens. While such molecules normally willonly bind two antigens (i.e. bispecific antibodies, BsAbs), antibodieswith additional specificities such as trispecific antibodies areencompassed by this expression when used herein. Examples of BsAbsinclude those with one arm directed against a tumor cell antigen and theother arm directed against a cytotoxic trigger molecule such asanti-FcÿRI/anti-CD15, anti-p185^(HER2) (CD16), anti-CD3/anti-malignantB-cell (ID10), anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97,anti-CD3/anti-renal cell carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1(anti-colon carcinoma), anti-CD3/anti-melanocyte stimulating hormoneanalog, anti-EGF receptor/anti-CD3, anti-CD3/anti-CAMA1,anti-CD3/anti-CD19, anti-CD3/MoV18, anti-neural cell ahesion molecule(NCAM)/anti-CD3, anti-folate binding protein (FBP)/anti-CD3, anti-pancarcinoma associated antigen (AMOC-31)/anti-CD3; BsAbs with one armwhich binds specifically to a tumor antigen and one arm which binds to atoxin such as anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-ÿ(IFN-ÿ)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcÿRI, FcÿRII or FcÿRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcÿR/anti-HIV; BsAbs for tumordetection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants (see Fanger et al., supra); and BsAbs as diagnostic tools suchas anti-rabbit IgG/anti-ferritin, anti-horse radish peroxidase(HRP)/anti-hormone, anti-somatostatin/anti-substance P,anti-HRP/anti-FITC, anti-CEA/anti-ÿ-galactosidase (see Nolan et al.,supra). Examples of trispecific antibodies includeanti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous protein(an “adhesin”, e.g. a receptor, ligand or enzyme) with the effectorfunctions of immunoglobulin constant domains. Structurally, theimmunoadhesins comprise a fusion of the adhesin amino acid sequence withthe desired binding specificity which is other than the antigenrecognition and binding site (antigen combining site) of an antibody(i.e. is “heterologous”) and an immunoglobulin constant domain sequence.The immunoglobulin constant domain sequence in the immunoadhesin may beobtained from any immunoglobulin, such as IgG₁, IgG₂, IgG₃, or IgG₄subtypes, IgA, IgE, IgD or IgM.

The term “ligand binding domain” as used herein refers to any nativecell-surface receptor or any region or derivative thereof retaining atleast a qualitative ligand binding ability, and preferably thebiological activity of a corresponding native receptor. In a specificembodiment, the receptor is from a cell-surface polypeptide having anextracellular domain which is homologous to a member of theimmunoglobulin supergenefamily. Other typical receptors, are not membersof the immunoglobulin supergenefamily but are nonetheless specificallycovered by this definition, are receptors for cytokines, and inparticular receptors with tyrosine kinase activity (receptor tyrosinekinases), members of the hematopoietin and nerve growth factor receptorsuperfamilies, and cell adhesion molecules, e.g. (E-, L- and P—)selectins.

The tent “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability, and preferably the biological activity of acorresponding native ligand. This definition, among others, specificallyincludes binding sequences from ligands for the above-mentionedreceptors.

As used herein the phrase “multispecific immunoadhesin” designatesimmunoadhesins (as hereinabove defined) having at least two bindingspecificities (i.e. combining two or more adhesin binding domains).Multispecific immunoadhesins can be assembled as heterodimers,heterotrimers or heterotetramers, essentially as disclosed in WO89/02922 (published 6 Apr. 1989), in EP 314,317 (published 3 May 1989),and in U.S. Pat. No. 5,116,964 issued 2 May 1992. Preferredmultispecific immunoadhesins are bispecific. Examples of bispecificimmunoadhesins include CD4-IgG/TNFreceptor-IgG andCD4-IgG/L-selectin-IgG. The last mentioned molecule combines the lymphnode binding function of the lymphocyte homing receptor (LHR,L-selectin), and the HIV binding function of CD4, and finds potentialapplication in the prevention or treatment of HIV infection, relatedconditions, or as a diagnostic.

An “antibody-immunoadhesin chimera (Ab/Ia chimera)” comprises a moleculewhich combines at least one binding domain of an antibody (as hereindefined) with at least one immunoadhesin (as defined in thisapplication). Exemplary Ab/Ia chimeras are the bispecific CD4-IgGchimeras described by Berg et al., supra and Chamow et al., supra.

The “interface” comprises those “contact” amino acid residues (or othernon-amino acid groups such as carbohydrate groups, NADH, biotin, FAD orhaem group) in the first polypeptide which interact with one or more“contact” amino acid residues (or other non-amino acid groups) in theinterface of the second polypeptide. The preferred interface is a domainof an immunoglobulin such as a variable domain or constant domain (orregions thereof), however the interface between the polypeptides forminga heteromultimeric receptor or the interface between two or more ligandssuch as NGF, NT-3 and BDNF are included within the scope of this term.The preferred interface comprises the C_(H)3 domain of an immunoglobulinwhich preferably is derived from an IgG antibody and most preferably anhuman IgG₁ antibody.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of the first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of the second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g. by alteringnucleic acid encoding the interface). Normally, nucleic acid encodingthe interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The upper limit for thenumber of original residues which are replaced is the total number ofresidues in the interface of the first polypeptide. The side chainvolumes of the various amino residues are shown in the following table.

TABLE 1 Properties of Amino Acid Residues Accessible One-Letter MASS^(a)VOLUME^(b) Surface Area^(c) Amino Acid Abbreviation (daltons) (Å³) (Å²)Alanine (Ala) A 71.08 88.6 115 Arginine (Arg) R 156.20 173.4 225Asparagine (Asn) N 114.11 117.7 160 Aspartic acid (Asp) D 115.09 111.1150 Cysteine (Cys) C 103.14 108.5 135 Glutamine (Gln) Q 128.14 143.9 180Glutamic acid (Glu) E 129.12 138.4 190 Glycine (Gly) G 57.06 60.1 75Histidine (His) H 137.15 153.2 195 Isoleucine (Ile) I 113.17 166.7 175Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18 168.6 200Methionine (Met) M 131.21 162.9 185 Phenylalinine (Phe) F 147.18 189.9210 Proline (Pro) P 97.12 122.7 145 Serine (Ser) S 87.08 89.0 115Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) W 186.21 227.8 255Tyrosine (Tyr) Y 163.18 193.6 230 Valine (Val) V 99.14 140.0 155^(a)Molecular weight amino acid minus that of water. Values fromHandbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical RubberPublishing Co., 1961. ^(b)Values from A. A. Zamyatnin, Prog. Biophys.Mol. Biol. 24:107-123, 1972. ^(c)Values from C. Chothia, J. Mol. Biol.105:1-14, 1975. The accessible surface area is defined in FIGS. 6-20 ofthis reference.

The preferred import residues for the formation of a protuberance aregenerally naturally occurring amino acid residues and are preferablyselected from arginine (R), phenylalanine (F), tyrosine (Y) andtryptophan (W). Most preferred are tryptophan and tyrosine. In thepreferred embodiment, the original residue for the formation of theprotuberance has a small side chain volume, such as alanine, asparagine,aspartic acid, glycine, serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of the second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface ofthe first polypeptide. The cavity may exist in the original interface ormay be introduced synthetically (e.g. by altering nucleic acid encodingthe interface). Normally, nucleic acid encoding the interface of thesecond polypeptide is altered to encode the cavity. To achieve this, thenucleic acid encoding at least one “original” amino acid residue in theinterface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue.The upper limit for the number of original residues which are replacedis the total number of residues in the interface of the secondpolypeptide. The side chain volumes of the various amino residues areshown in Table 1 above. The preferred import residues for the formationof a cavity are usually naturally occurring amino acid residues and arepreferably selected from alanine (A), serine (S), threonine (T) andvaline (V). Most preferred are serine, alanine or threonine. In thepreferred embodiment, the original residue for the formation of theprotuberance has a large side chain volume, such as tyrosine, arginine,phenylalanine or tryptophan.

A “original” amino acid residue is one which is replaced by an “import”residue which can have a smaller or larger side chain volume than theoriginal residue. The import amino acid residue can be a naturallyoccurring or non-naturally occurring amino acid residue, but preferablyis the former. “Naturally occurring” amino acid residues are thoseresidues encoded by the genetic code and listed in Table 1 above. By“non-naturally occurring” amino acid residue is meant a residue which isnot encoded by the genetic code, but which is able to covalently bindadjacent amino acid residue(s) in the polypeptide chain. Examples ofnon-naturally occurring amino acid residues are norleucine, ornithine,norvaline, homoserine and other amino acid residue analogues such asthose described in Ellman et al., Meth. Enzym. 202:301-336 (1991), forexample. To generate such non-naturally occurring amino acid residues,the procedures of Noren et al. Science 244: 182 (1989) and Ellman etal., supra can be used. Briefly, this involves chemically activating asuppressor tRNA with a non-naturally occurring amino acid residuefollowed by in vitro transcription and translation of the RNA. Themethod of the instant invention involves replacing at least one originalamino acid residue, but more than one original residue can be replaced.Normally, no more than the total residues in the interface of the firstor second polypeptide will comprise original amino acid residues whichare replaced. The preferred original residues for replacement are“buried”. By “buried” is meant that the residue is essentiallyinaccessible to solvent. The preferred import residue is not cysteine toprevent possible oxidation or mispairing of disulfide bonds.

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of thefirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity relies on modeling the protuberance/cavity pair based upon athree-dimensional structure such as that obtained by X-raycrystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

By “original nucleic acid” is meant the nucleic acid encoding apolypeptide of interest which can be “altered” (i.e. geneticallyengineered or mutated) to encode a protuberance or cavity. The originalor starting nucleic acid may be a naturally occurring nucleic acid ormay comprise a nucleic acid which has been subjected to prior alteration(e.g. a humanized antibody fragment). By “altering” the nucleic acid ismeant that the original nucleic acid is mutated by inserting, deletingor replacing at least one codon encoding an amino acid residue ofinterest. Normally, a codon encoding an original residue is replaced bya codon encoding an import residue. Techniques for genetically modifyinga DNA in this manner have been reviewed in Mutagenesis: a PracticalApproach, M. J. McPherson, Ed., (IRL Press, Oxford, UK. (1991), andinclude site-directed mutagenesis, cassette mutagenesis and polymerasechain reaction (PCR) mutagenesis, for example.

The protuberance or cavity can be “introduced” into the interface of thefirst or second polypeptide by synthetic means, e.g. by recombinanttechniques, in vitro peptide synthesis, those techniques for introducingnon-naturally occurring amino acid residues previously described, byenzymatic or chemical coupling of peptides or some combination of thesetechniques. According, the protuberance or cavity which is “introduced”is “non-naturally occurring” or “non-native”, which means that it doesnot exist in nature or in the original polypeptide (e.g. a humanizedmonoclonal antibody).

Preferably the import amino acid residue for forming the protuberancehas a relatively small number of “rotamers” (e.g. about 3-6). A“rotomer” is an energetically favorable conformation of an amino acidside chain. The number of rotomers of the various amino acid residuesare reviewed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987).

“Isolated” heteromultimer means heteromultimer which has been identifiedand separated and/or recovered from a component of its natural cellculture environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the heteromultimer, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, theheteromultimer will be purified (1) to greater than 95% by weight ofprotein as determined by the Lowry method, and most preferably more than99% by weight, (2) to a degree sufficient to obtain at least 15 residuesof N-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain.

The heteromultimers of the present invention are generally purified tosubstantial homogeneity. The phrases “substantially homogeneous”,“substantially homogeneous form” and “substantial homogeneity” are usedto indicate that the product is substantially devoid of by-productsoriginated from undesired polypeptide combinations (e.g. homomultimers).Expressed in terms of purity, substantial homogeneity means that theamount of by-products does not exceed 10%, and preferably is below 5%,more preferably below 1%, most preferably below 0.5%, wherein thepercentages are by weight.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

II. PREPARATION OF THE HETEROMULTIMER

1. Preparation of the Starting Materials

As a first step, the first and second polypeptide (and any additionalpolypeptides forming the heteromultimer) are selected. Normally, thenucleic acid encoding these polypeptides needs to be isolated so that itcan be altered to encode the protuberance or cavity, or both, as hereindefined. However, the mutations can be introduced using synthetic means,e.g. by using a peptide synthesizer. Also, in the case where the importresidue is a non-naturally occurring residue, the method of Noren etal., supra is available for making polypeptides having suchsubstitutions. Additionally, part of the heteromultimer is suitably maderecombinantly in cell culture and other part(s) of the molecule are madeby those techniques mentioned above.

Techniques for isolating antibodies and preparing immunoadhesins follow.However, it will be appreciated that the heteromultimer can be formedfrom, or incorporate, other polypeptides using techniques which areknown in the art. For example, nucleic acid encoding a polypeptide ofinterest (e.g. a ligand, receptor or enzyme) can be isolated from a cDNAlibrary prepared from tissue believed to possess the polypeptide mRNAand to express it at a detectable level. Libraries are screened withprobes (such as antibodies or oligonucleotides of about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989).

(i) Antibody Preparation

Several techniques for the production of antibodies have been describedwhich include the traditional hybridoma method for making monoclonalantibodies, recombinant techniques for making antibodies (includingchimeric antibodies, e.g. humanized antibodies), antibody production intransgenic animals and the recently described phage display technologyfor preparing “fully human” antibodies. These techniques shall bedescribed briefly below.

Polyclonal antibodies to the antigen of interest generally can be raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the antigen and an adjuvant. It may be useful to conjugatethe antigen (or a fragment containing the target amino acid sequence) toa protein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example maleimidobenzoyl sulfosuccinimide ester (conjugation throughcysteine residues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups. Animals are immunized against theimmunogenic conjugates or derivatives by combining 1 mg of 1 ÿg ofconjugate (for rabbits or mice, respectively) with 3 volumes of Freud'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of conjugate in Freud's complete adjuvant bysubcutaneous injection at multiple sites. 7 to 14 days later the animalsare bled and the serum is assayed for antibody titer. Animals areboosted until the titer plateaus. Preferably, the animal is boosted withthe conjugate of the same antigen, but conjugated to a different proteinand/or through a different cross-linking reagent. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies using the hybridoma method first described byKohler & Milstein, Nature 256:495 (1975) or may be made by recombinantDNA methods (Cabilly et al., U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as hamster, isimmunized as hereinabove described to elicit lymphocytes that produce,or are capable of producing, antibodies that will specifically bind tothe protein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 [Academic Press, 1986]). The hybridoma cells thus preparedare seeded and grown in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, parental myeloma cells. For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which substances prevent the growth of HGPRT-deficient cells.Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 [1984]; and Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63, MarcelDekker, Inc., New York, 1987). See, also, Boerner et al., J. Immunol.,147(1):86-95 (1991) and WO 91/17769, published Nov. 28, 1991, fortechniques for the production of human monoclonal antibodies. Culturemedium in which hybridoma cells are growing is assayed for production ofmonoclonal antibodies directed against the antigen of interest.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson & Pollard, Anal. Biochem. 107:220 (1980). After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity, and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods. Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-104 (Academic Press, 1986).Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium or RPMI-1640 medium. In addition, the hybridomacells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Alternatively, it is now possible to produce transgenic animals (e.g.mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-255(1993) and Jakobovits et al., Nature 362:255-258 (1993).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), using theantigen of interest to select for a suitable antibody or antibodyfragment. Clackson et al., Nature, 352:624-628 (1991) and Marks et al.,J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Mark et al., Bio/Technol. 10:779-783[1992]), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nuc. Acids Res., 21:2265-2266 [1993]). Thus, these techniques areviable alternatives to traditional monoclonal antibody hybridomatechniques for isolation of “monoclonal” antibodies (especially humanantibodies) which are encompassed by the present invention.

DNA encoding the antibodies of the invention is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences, Morrisonet al., Proc. Nat. Acad. Sci. 81:6851 (1984). In that manner, “chimeric”antibodies are prepared that have the binding specificity of ananti-antigen monoclonal antibody herein.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 [1986]; Riechmann et al., Nature332:323-327 [1988]; Verhoeyen et al., Science 239:1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues,and possibly some FR residues, are substituted by residues fromanalogous sites in rodent antibodies. It is important that antibodies behumanized with retention of high affinity for the antigen and otherfavorable biological properties. To achieve this goal, according to apreferred method, humanized antibodies are prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three dimensional models of the parental and humanizedsequences. Three dimensional immunoglobulin models are familiar to thoseskilled in the art. Computer programs are available which illustrate anddisplay probable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. For further details see WO 92/22653, published Dec. 23, 1992.

(ii) Immunoadhesin Preparation

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982);EP 120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Köhler etal., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res.41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984);Morrison, Science 229:1202 (1985); Morrison et al., Proc. Natl. Acad.Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559.Reassorted immunoglobulin chains also are known. See, for example, U.S.Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references citedtherein.

Chimeras constructed from an adhesin binding domain sequence linked toan appropriate immunoglobulin constant domain sequence (immunoadhesins)are known in the art. Immunoadhesins reported in the literature includefusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci.USA 84:2936-2940 [1987]); CD4 (Capon et al., Nature 337:525-531 [1989];Traunecker et al., Nature 339:68-70 [1989]; Zettmeissl et al., DNA CellBiol. USA 9:347-353 [1990]; and Byrn et al., Nature 344:667-670 [1990]);L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 [1990]; and Watson et al., Nature 349:164-167 [1991]);CD44 (Aruffo et al., Cell 61:1303-1313 [1990]); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 [1991]); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 [1991]); CD22 (Stamenkovic et al., Cell 66:1133-1144[1991]); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 [1991]; Lesslauer et al., Eur. J. Immunol. 27:2883-2886[1991]; and Peppel et al., J. Exp. Med. 174:1483-1489 [1991]); and IgEreceptor ÿ (Ridgway and Gorman. J. Cell. Biol. Vol. 115, Abstract No.1448 [1991]).

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain [ECD] ofa receptor) with the hinge and Fc regions of an immunoglobulin heavychain (see FIG. 3). Ordinarily, when preparing the immunoadhesins of thepresent invention, nucleic acid encoding the binding domain of theadhesin will be fused C-terminally to nucleic acid encoding theN-terminus of an immunoglobulin constant domain sequence, howeverN-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the Ia.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc domain of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG₁, IgG₂, orIgG₃ heavy chain. The precise site at which the fusion is made is notcritical, and the optimal site can be determined by routineexperimentation.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

(a) AC_(L-)AC_(L);

(b) AC_(H-)[AC_(H), AC_(L-)AC_(H), AC_(L)V_(H)C_(H), orV_(L)C_(L)AC_(H)];

(c) AC_(L-)AC_(H-)[AC_(L-)AC_(H), AC_(L-)V_(H)C_(H), V_(L)C_(L-)AC_(H),or V_(L)C_(L)V_(H)C_(H)];

(d) AC_(L-)V_(H)C_(H-)[AC_(H), or AC_(L-)V_(H)C_(H), orV_(L)C_(L-)AC_(H)];

(e) V_(L)C_(L)AC_(H-)[AC_(L-)V_(H)C_(H), or V_(L)C_(L)AC_(H)]; and

(f) [A-Y]_(n-)[V_(L)C_(L-)V_(H)C_(H)]₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, etal., Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG₁ and IgG₃ immunoglobulin sequencesis preferred. A major advantage of using IgG₁ is that IgG₁immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG₃ requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG₃ hingeis longer and more flexible, so it can accommodate larger “adhesin”domains that may not fold or function properly when fused to IgG₁.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG₁, IgG₂ and IgG₄ all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG₄ does not activate complement,and IgG₂ is significantly weaker at complement activation than IgG₁.Moreover, unlike IgG₁, IgG₂ does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG₃ is optimal for complement activation,its in vivo half-life is approximately one third of the other IgGisotypes. Another important consideration for immunoadhesins designed tobe used as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG₁ hasonly four serologically-defined allotypic sites, two of which (G1m and2) are located in the Fc region; and one of these sites, G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic.Thus, the potential immunogenicity of a ÿ3 immunoadhesin is greater thanthat of a ÿ1 immunoadhesin.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an Ig cDNA sequence.However, fusion to genomic Ig fragments can also be used (see, e.g.Gascoigne et al., supra; Aruffo et al., Cell 61:1303-1313 [1990]; andStamenkovic et al., Cell 66:1133-1144 [1991]). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and the Igparts of the immunoadhesin are inserted in tandem into a plasmid vectorthat directs efficient expression in the chosen host cells.

2. Generating a Protuberance and/or Cavity

As a first step to selecting original residues for forming theprotuberance and/or cavity, the three-dimensional structure of theheteromultimer is obtained using techniques which are well known in theart such as X-ray crystallography or NMR. Based on the three-dimensionalstructure, those skilled in the art will be able to identify theinterface residues.

The preferred interface is the C_(H)3 domain of an immunoglobulinconstant domain. The interface residues of the C_(H)3 domains of IgG,IgA, IgD, IgE and IgM are identified in FIG. 5, including those whichare optimal for replacing with import residues. The interface residuesof various IgG subtypes are illustrated in FIG. 6. “Buried” residues arealso identified. The basis for engineering the C_(H)3 interface is thatX-ray crystallography has demonstrated that the intermolecularassociation between human IgG₁ heavy chains in the Fc region includesextensive protein/protein interaction between C_(H)3 domains whereas theglycosylated C_(H)2 domains interact via their carbohydrate(Deisenhofer, Biochem. 20:2361 [1981]). In addition there are twointer-heavy chain disulfide bonds which are efficiently formed duringantibody expression in mammalian cells unless the heavy chain istruncated to remove C_(H)2 and C_(H)3 domains (King et al., Biochem. J.281:317 [1992]). Thus, heavy chain assembly appears to promote disulfidebond formation rather than vice versa. Taken together these structuraland functional data led to the hypothesis that antibody heavy chainassociation is directed by the C_(H)3 domains. It was further speculatedthat the interface between C_(H)3 domains might be engineered to promoteformation of heteromultimers of different heavy chains and hinderassembly of corresponding homomultimers. The experiments describedherein demonstrated that it was possible to promote the formation ofheteromultimers over homomultimers using this approach. Thus, it ispossible to generate a polypeptide fusion comprising a polypeptide ofinterest and the C_(H)3 domain of an antibody to form a first or secondpolypeptide. The preferred C_(H)3 domain is derived from an IgGantibody, such as an human IgG₁. The interface residues of human IgG₁are depicted in FIG. 7.

Those interface residues which can potentially constitute candidates forforming the protuberance or cavity are identified. It is preferable toselect “buried” residues to be replaced. To determine whether a residueis buried, the surface accessibility program of Lee et al. J. Mol. Biol.55: 379-400 (1971) can be used to calculate the solvent accessibility(SA) of residues in the interface. Then, the SA for the residues of eachof the first and second polypeptide can be separately calculated afterremoval of the other polypeptide. The difference in SA of each residuebetween the monomer and dimer forms of the interface can then becalculated by: SA (dimer)-SA (monomer). This provides a list of residueswhich lose SA on formation of the dimer. The SA of each residue in thedimer is compared to the theoretical SA of the same amino acid in thetripeptide Gly-X-Gly, where X=the amino acid of interest (Rose et al.Science 229: 834-838 [1985]). Residues which (a) lost SA in the dimercompared to the monomer and (b) had an SA less than 26% of that in theircorresponding tripeptide are considered as interface residues. Twocategories may be delineated: those which have an SA<10% compared totheir corresponding tripeptide (i.e. “buried”) and those which have25%>SA>10% compared to their corresponding tripeptide (i.e. “partiallyburied”).

TABLE 2 SA Lost Monomer → 4 Dimer % Tripeptide Residue Poly- Poly- Poly-Poly- No.^(†) peptide A peptide B peptide A peptide B Q347 22.1 31.025.0 26.5 Y349 79.8 83.9 5.2 5.7 L351 67.4 77.7 3.9 2.0 S354 53.4 52.811.3 11.7 E357 43.7 45.3 0.4 1.3 S364 21.5 15.1 0.5 1.4 T366 29.3 25.80.0 0.1 L368 25.5 29.7 1.0 1.1 K370 55.8 62.3 11.5 11.0 T394 64.0 58.50.6 1.4 V397 50.3 49.5 13.2 11.0 D399 39.7 33.7 5.7 5.7 F405 53.7 52.10.0 0.0 Y407 89.1 90.3 0.0 0.0 K409 86.8 92.3 0.7 0.6 T411 4.3 7.5 12.79.8 ^(†)residue numbering as in IgG crystal structure (Deisenhofer,Biochemistry 20:2361-2370 [1981]).

The effect of replacing residues on the polypeptide chain structure canbe studied using a molecular graphics modeling program such as theInsight™ program (Biosym Technologies). Using the program, those buriedresidues in the interface of the first polypeptide which have a smallside chain volume can be changed to residues having a larger side chainvolume (i.e. a protuberance), for example. Then, the residues in theinterface of the second polypeptide which are in proximity to theprotuberance are examined to find a suitable residue for forming thecavity. Normally, this residue will have a large side chain volume andis replaced with a residue having a smaller side chain volume. Incertain embodiments, examination of the three-dimensional structure ofthe interface will reveal a suitably positioned and dimensionedprotuberance on the interface of the first polypeptide or a cavity onthe interface of the second polypeptide. In these instances, it is onlynecessary to model a single mutant, i.e., with a syntheticallyintroduced protuberance or cavity.

With respect to selecting potential original residues for replacementwhere the first and second polypeptide each comprise a C_(H)3 domain,the C_(H)3/C_(H)3 interface of human IgG₁ involves sixteen residues oneach domain located on four anti-parallel ÿ-strands which buries 1090 Å²from each surface (Deisenhofer, supra) and Miller, J. Mol. Biol. 216:965[1990]). See FIG. 7 herein. Mutations are preferably targeted toresidues located on the two central anti-parallel ÿ-strands. The aim isto minimize the risk that the protuberances which are created can beaccommodated by protruding into surrounding solvent rather than bycompensatory cavities in the partner C_(H)3 domain.

Once the preferred original/import residues are identified by molecularmodeling, the amino acid replacements are introduced into thepolypeptide using techniques which are well known in the art. Normallythe DNA encoding the polypeptide is genetically engineered using thetechniques described in Mutagenesis: a Practical Approach, supra.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution variants of the DNA encoding the first or secondpolypeptide. This technique is well known in the art as described byAdelman et al., DNA, 2:183 (1983). Briefly, first or second polypeptideDNA is altered by hybridizing an oligonucleotide encoding the desiredmutation to a DNA template, where the template is the single-strandedform of a plasmid or bacteriophage containing the unaltered or nativeDNA sequence of heteromultimer. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat will thus incorporate the oligonucleotide primer, and will code forthe selected alteration in the heteromultimer DNA.

Cassette mutagenesis can be performed as described Wells et al. Gene34:315 (1985) by replacing a region of the DNA of interest with asynthetic mutant fragment generated by annealing complimentaryoligonucleotides. PCR mutagenesis is also suitable for making variantsof the first or second polypeptide DNA. While the following discussionrefers to DNA, it is understood that the technique also findsapplication with RNA. The PCR technique generally refers to thefollowing procedure (see Erlich, Science, 252:1643-1650 [1991], thechapter by R. Higuchi, p. 61-70).

This invention also encompasses, in addition to the protuberance orcavity mutations, amino acid sequence variants of the heteromultimerwhich can be prepared by introducing appropriate nucleotide changes intothe heteromultimer DNA, or by synthesis of the desired heteromultimerpolypeptide. Such variants include, for example, deletions from, orinsertions or substitutions of, residues within the amino acid sequencesof the first and second polypeptides forming the heteromultimer. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired antigen-binding characteristics. The amino acid changes also mayalter post-translational processes of the heteromultimer, such aschanging the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of theheteromultimer polypeptides that are preferred locations for mutagenesisis called “alanine scanning mutagenesis,” as described by Cunningham andWells, Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g. charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the surrounding aqueous environment in or outsidethe cell. Those domains demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined.

Normally the mutations will involve conservative amino acid replacementsin non-functional regions of the heteromultimer. Exemplary mutations areshown in the following table.

TABLE 3 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; met;ile ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F)leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) serser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; ala; leu norleucine

Covalent modifications of the heteromultimer polypeptides are includedwithin the scope of this invention. Covalent modifications of theheteromultimer can be introduced into the molecule by reacting targetedamino acid residues of the heteromultimer or fragments thereof with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues. Another type of covalentmodification of the heteromultimer polypeptide included within the scopeof this invention comprises altering the native glycosylation pattern ofthe polypeptide. By altering is meant deleting one or more carbohydratemoieties found in the original heteromultimer, and/or adding one or moreglycosylation sites that are not present in the original heteromultimer.Addition of glycosylation sites to the heteromultimer polypeptide isconveniently accomplished by altering the amino acid sequence such thatit contains one or more N-linked glycosylation sites. The alteration mayalso be made by the addition of, or substitution by, one or more serineor threonine residues to the original heteromultimer sequence (forO-linked glycosylation sites). For ease, the heteromultimer amino acidsequence is preferably altered through changes at the DNA level,particularly by mutating the DNA encoding the heteromultimer polypeptideat preselected bases such that codons are generated that will translateinto the desired amino acids. Another means of increasing the number ofcarbohydrate moieties on the heteromultimer polypeptide is by chemicalor enzymatic coupling of glycosides to the polypeptide. These methodsare described in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). Removal ofcarbohydrate moieties present on the heteromultimer may be accomplishedchemically or enzymatically.

Another type of covalent modification of heteromultimer compriseslinking the heteromultimer polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Since it is often difficult to predict in advance the characteristics ofa variant heteromultimer, it will be appreciated that some screening ofthe recovered variant will be needed to select the optimal variant.

3. Expression of the Heteromultimer

Following mutation of the DNA as discussed in the preceding section, theDNA encoding the molecule is expressed using recombinant techniqueswhich are widely available in the art. Often, the expression system ofchoice will involve a mammalian cell expression vector and host so thatthe heteromultimer is appropriately glycosylated (e.g. in the case ofheteromultimers comprising antibody domains which are glycosylated).However, the molecules can also be produced in the prokaryoticexpression systems elaborated below. Normally, the host cell will betransformed with DNA encoding both the first polypeptide and the secondpolypeptide and other polypeptide(s) required to form theheteromultimer, on a single vector or independent vectors. However, itis possible to express the first polypeptide and second polypeptide inindependent expression systems and couple the expressed polypeptides invitro.

The nucleic acid (e.g., cDNA or genomic DNA) encoding the heteromultimeris inserted into a replicable vector for further cloning (amplificationof the DNA) or for expression. Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

The polypeptides of the heteromultimer may be produced as fusionpolypeptides with a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the DNA that is inserted into the vector.The heterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells, the signal sequence may besubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,alpha factor leader (including Saccharomyces and Kluyveromyces ÿ-factorleaders, the latter described in U.S. Pat. No. 5,010,182 issued 23 Apr.1991), or acid phosphatase leader, the C. albicans glucoamylase leader(EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression the nativesignal sequence (e.g., the antibody or adhesin presequence that normallydirects secretion of these molecules from human cells in vivo) issatisfactory, although other mammalian signal sequences may be suitableas well as viral secretory leaders, for example, the herpes simplex gDsignal. The DNA for such precursor region is ligated in reading frame toDNA encoding the polypeptides forming the heteromultimer.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2ÿ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet. 1:327[1982]), mycophenolic acid (Mulligan et al., Science 209:1422 [1980]) orhygromycin (Sugden et al., Mol. Cell. Biol. 5:410-413 [1985]). The threeexamples given above employ bacterial genes under eukaryotic control toconvey resistance to the appropriate drug G418 or neomycin (geneticin),xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theheteromultimer nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesheteromultimer. Increased quantities of heteromultimer are synthesizedfrom the amplified DNA. Other examples of amplifiable genes includemetallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77:4216 (1980). The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding thecomponents of the heteromultimer This amplification technique can beused with any otherwise suitable host, 24 e.g., ATCC No. CCL61 CHO-K1,notwithstanding the presence of endogenous DHFR if, for example, amutant DHFR gene that is highly resistant to Mtx is employed (EP117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding heteromultimer, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature 282:39 [1979];Kingsman et al., Gene 7:141 [1979]; or Tschemper et al., Gene 10:157[1980]). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics 85:12 [1977]). The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 ÿm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Bianchi et al.,Curr. Genet. 12:185 (1987). More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis. Van den Berg, Bio/Technology 8:135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., Bio/Technology 9:968-975 (1991).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theheteromultimer nucleic acid. A large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to heteromultimer-encoding DNA by removing the promoterfrom the source DNA by restriction enzyme digestion and inserting theisolated promoter sequence into the vector.

Promoters suitable for use with prokaryotic hosts include theÿ-lactamase and lactose promoter systems (Chang et al., Nature 275:615[1978]; and Goeddel et al., Nature 281:544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 [1980] and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 [1983]).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the heteromultimer (Siebenlistet al., Cell 20:269 [1980]) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the heteromultimer.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073 [1980]) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg. 7:149 [1968]; and Holland, Biochemistry 17:4900[1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Heteromultimer transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand most preferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter orfrom heat-shock promoters.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature 273:113 (1978); Mulligan and Berg,Science 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA 78:7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene 18:355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature 297:598-601 (1982) on expressionof human ÿ-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg.Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of thehuman interferon ÿ1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

Transcription of DNA encoding the heteromultimer by higher eukaryotes isoften increased by inserting an enhancer sequence into the vector.Enhancers are relatively orientation and position independent, havingbeen found 5′ (Laimins et al., Proc. Natl. Acad. Sci. USA 78:993 [1981])and 3′ (Lusky et al., Mol. Cell Bio. 3:1108 [1983]) to the transcriptionunit, within an intron (Banerji et al., Cell 33:729 [1983]), as well aswithin the coding sequence itself (Osborne et al., Mol. Cell Bio. 4:1293[1984]). Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, ÿ-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the heteromultimer-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the heteromultimer.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology 65:499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding heteromultimer. In general, transient expression involvesthe use of an expression vector that is able to replicate efficiently ina host cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Sambrook et al., supra,pp. 16.17-16.22. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of heteromultimers having desired bindingspecificities/affinities.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the heteromultimer in recombinant vertebrate cell cultureare described in Gething et al., Nature 293:620-625 (1981); Mantei etal., Nature 281:40-46 (1979); Levinson et al.; EP 117,060; and EP117,058. A particularly useful plasmid for mammalian cell cultureexpression of the heteromultimer is pRK5 (EP 307,247) or pSVI6B (PCTpub. no. WO 91/08291 published 13 Jun. 1991).

The choice of host cell line for the expression of heteromultimerdepends mainly on the expression vector. Another consideration is theamount of protein that is required. Milligram quantities often can beproduced by transient transfections. For example, the adenovirusEIA-transformed 293 human embryonic kidney cell line can be transfectedtransiently with pRK5-based vectors by a modification of the calciumphosphate method to allow efficient heteromultimer expression.CDM8-based vectors can be used to transfect COS cells by theDEAE-dextran method (Aruffo et al., Cell 61:1303-1313 [1990]; andZettmeissl et al., DNA Cell Biol. (US) 9:347-353 [1990]). If largeramounts of protein are desired, the immunoadhesin can be expressed afterstable transfection of a host cell line. For example, a pRK5-basedvector can be introduced into Chinese hamster ovary (CHO) cells in thepresence of an additional plasmid encoding dihydrofolate reductase(DHFR) and conferring resistance to G418. Clones resistant to G418 canbe selected in culture. These clones are grown in the presence ofincreasing levels of DHFR inhibitor methotrexate and clones are selectedin which the number of gene copies encoding the DHFR and heteromultimersequences is co-amplified. If the immunoadhesin contains a hydrophobicleader sequence at its N-terminus, it is likely to be processed andsecreted by the transfected cells. The expression of immunoadhesins withmore complex structures may require uniquely suited host cells. Forexample, components such as light chain or J chain may be provided bycertain myeloma or hybridoma host cells (Gascoigne et al., supra; andMartin et al., J. Virol. 67:3561-3568 [1993]).

Other suitable host cells for cloning or expressing the vectors hereinare prokaryote, yeast, or other higher eukaryote cells described above.Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 is aparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell should secrete minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strain 27C7. The complete genotype of 27C7 is tonAÿ ptr3 phoAÿE15ÿ(argF-lac)169 ompTÿ degP41kan^(r). Strain 27C7 was deposited on 30 Oct.1991 in the American Type Culture Collection as ATCC No. 55,244.Alternatively, the strain of E. coli having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990 may be employed.Alternatively, methods of cloning, e.g., PCR or other nucleic acidpolymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forheteromultimer-encoding vectors. Saccharomyces cerevisiae, or commonbaker's yeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe (Beach and Nurse, Nature 290:140 [1981]; EP 139,383 published May2, 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,supra) such as, e.g., K. lactis [MW98-8C, CBS683, CBS4574; Louvencourtet al., J. Bacteriol., 737 (1983)], K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., supra), K.thermotolerans, and K. marxianus; yarrowia [EP 402,226]; Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol. 28:265-278 [1988]);Candida; Trichoderma reesia [EP 244,234]; Neurospora crassa (Case etal., Proc. Natl. Acad. Sci. USA 76:5259-5263 [1979]); Schwanniomycessuch as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillushosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.Commun. 112:284-289 [1983]; Tilburn et al., Gene 26:205-221 [1983];Yelton et al., Proc. Natl. Acad. Sci. USA 81:1470-1474 [1984]) and A.niger (Kelly and Hynes, EMBO J. 4:475-479 [1985]).

Suitable host cells for the expression of glycosylated heteromultimerare derived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology 6:47-55(1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature315:592-594 (1985). A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the heteromultimer DNA. During incubation of the plant cellculture with A. tumefaciens, the DNA encoding the heteromultimer istransferred to the plant cell host such that it is transfected, andwill, under appropriate conditions, express the heteromultimer DNA. Inaddition, regulatory and signal sequences compatible with plant cellsare available, such as the nopaline synthase promoter andpolyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen.1:561 (1982). In addition, DNA segments isolated from the upstreamregion of the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. EP 321,196 published 21 Jun. 1989.

The preferred hosts are vertebrate cells, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure inrecent years (Tissue Culture, Academic Press, Kruse and Patterson,editors [1973]). Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216[1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251[1980]); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected with the above-described expression orcloning vectors of this invention and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.Depending on the host cell used, transfection is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described in section 1.82 of Sambrook et al.,supra, or electroporation is generally used for prokaryotes or othercells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene 23:315 (1983) and WO 89/05859published 29 Jun. 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued 16 Aug. 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact. 130:946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) 76:3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, etc.,may also be used. For various techniques for transforming mammaliancells, see Keown et al., Methods in Enzymology (1989), Keown et al.,Methods in Enzymology 185:527-537 (1990), and Mansour et al., Nature336:348-352 (1988).

Prokaryotic cells used to produce the heteromultimer polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the heteromultimer of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM],Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium([DMEM], Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz. 58:44 (1979),Barnes and Sato, Anal. Biochem. 102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of allof which are incorporated herein by reference, may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed., IRLPress, 1991.

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

4. Recovery of the Heteromultimer

The heteromultimer preferably is generally recovered from the culturemedium as a secreted polypeptide, although it also may be recovered fromhost cell lysate when directly produced without a secretory signal. Ifthe heteromultimer is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100)

When the heteromultimer is produced in a recombinant cell other than oneof human origin, it is completely free of proteins or polypeptides ofhuman origin. However, it is necessary to purify the heteromultimer fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to heteromultimer. As a first step, theculture medium or lysate is normally centrifuged to remove particulatecell debris.

Heterodimers having antibody constant domains can be convenientlypurified by hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography, with affinity chromatography beingthe preferred purification technique. Where the heteromultimer comprisesa C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg,N.J.) is useful for purification. Other techniques for proteinpurification such as fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin Sepharose, chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the polypeptide to be recovered. The suitabilityof protein A as an affinity ligand depends on the species and isotype ofthe immunoglobulin Fc domain that is used in the chimera. Protein A canbe used to purify immunoadhesins that are based on human ÿ1, ÿ2, or ÿ4heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 [1983]).Protein G is recommended for all mouse isotypes and for human ÿ3 (Gusset al., EMBO J. 5:15671575 [1986]). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. The conditions forbinding an immunoadhesin to the protein A or G affinity column aredictated entirely by the characteristics of the Fc domain; that is, itsspecies and isotype. Generally, when the proper ligand is chosen,efficient binding occurs directly from unconditioned culture fluid. Onedistinguishing feature of immunoadhesins is that, for human ÿ1molecules, the binding capacity for protein A is somewhat diminishedrelative to an antibody of the same Fc type. Bound immunoadhesin can beefficiently eluted either at acidic pH (at or above 3.0), or in aneutral pH buffer containing a mildly chaotropic salt. This affinitychromatography step can result in a heterodimer preparation that is >95%pure.

5. Uses for the Heteromultimer

Many therapeutic applications for the heteromultimer are contemplated.For example, the heteromultimer can be used for redirected cytotoxicity(e.g. to kill tumor cells), as a vaccine adjuvant, for deliveringthrombolytic agents to clots, for delivering immunotoxins to tumorcells, for converting enzyme activated prodrugs at a target site (e.g. atumor), for treating infectious diseases or targeting immune complexesto cell surface receptors.

Therapeutic formulations of the heteromultimer are prepared for storageby mixing the heteromultimer having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,[1980]), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

The heteromultimer also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate]microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

The heteromultimer to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The heteromultimer ordinarily will be stored inlyophilized form or in solution.

Therapeutic heteromultimer compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The route of heteromultimer administration is in accord with knownmethods, e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems as noted below.The heteromultimer is administered continuously by infusion or by bolusinjection.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981) andLanger, Chem. Tech. 12:98-105 (1982) or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers22:547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release heteromultimer compositions also include liposomallyentrapped heteromultimer. Liposomes containing heteromultimer areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal heteromultimer therapy.

An effective amount of heteromultimer to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 ÿg/kg to up to 10 mg/kgor more, depending on the factors mentioned above. Typically, theclinician will administer heteromultimer until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays.

The heteromultimers described herein can also be used in enzymeimmunoassays. To achieve this, one arm of the heteromultimer can bedesigned to bind to a specific epitope on the enzyme so that bindingdoes not cause enzyme inhibition, the other arm of the heteromultimercan be designed to bind to the immobilizing matrix ensuring a highenzyme density at the desired site. Examples of such diagnosticheteromultimers include those having specificity for IgG as well asferritin, and those having binding specificities for horse radishperoxidase (HRP) as well as a hormone, for example.

The heteromultimers can be designed for use in two-site immunoassays.For example, two bispecific heteromultimers are produced binding to twoseparate epitopes on the analyte protein—one heteromultimer binds thecomplex to an insoluble matrix, the other binds an indicator enzyme.

Heteromultimers can also be used for in vitro or in vivo immunodiagnosisof various diseases such as cancer. To facilitate this diagnostic use,one arm of the heteromultimer can be designed to bind a tumor associatedantigen and the other arm can bind a detectable marker (e.g. a chelatorwhich binds a radionuclide). For example, a heteromultimer havingspecificities for the tumor associated antigen CEA as well as a bivalenthapten can be used for imaging of colorectal and thryroid carcinomas.Other non-therapeutic, diagnostic uses for the heteromultimer will beapparent to the skilled practitioner.

For diagnostic applications, at least one arm of the heteromultimertypically will be labeled directly or indirectly with a detectablemoiety. The detectable moiety can be any one which is capable ofproducing, either directly or indirectly, a detectable signal. Forexample, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C,³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, suchas alkaline phosphatase, beta-galactosidase or horseradish peroxidase(HRP).

Any method known in the art for separately conjugating theheteromultimer to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature 144:945 (1962); David et al.,Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

The heteromultimers of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof heteromultimer. The amount of analyte in the test sample is inverselyproportional to the amount of standard that becomes bound to theheteromultimer. To facilitate determining the amount of standard thatbecomes bound, the heteromultimers generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the heteromultimers may conveniently be separated from the standardand analyte which remain unbound.

The heteromultimers are particularly useful for sandwich assays whichinvolve the use of two molecules, each capable of binding to a differentimmunogenic portion, or epitope, of the sample to be detected. In asandwich assay, the test sample analyte is bound by a first arm of theheteromultimer which is immobilized on a solid support, and thereafter asecond arm of the heteromultimer binds to the analyte, thus forming aninsoluble three part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond arm of the heteromultimer may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

EXAMPLES

The C_(H)3 interface between the humanized anti-CD3/CD4-IgG chimerapreviously described by Chamow et al. J. Immunol. 153:4268 (1994) wasengineered to maximize the percentage of heteromultimers which could berecovered. Protuberance-into-cavity and wild-type C_(H)3 variants werecompared in their ability to direct the formation of a humanizedantibody-immunoadhesin chimera (Ab/Ia) anti-CD3/CD4-IgG.

Thus, mutations were constructed in the C_(H)3 domain of the humanizedanti-CD3 antibody heavy chain and in CD4-IgG by site-directedmutagenesis using mismatched oligonucleotides (Kunkel et al., MethodsEnzymol. 154: 367 [1987] and P. Carter, in Mutagenesis: a PracticalApproach, M. J. McPherson, Ed., IRL Press, Oxford, UK, pp. 1-25 [1991])and verified by dideoxynucleotide sequencing (Sanger et al., Proc. Natl.Acad. Sci. USA 74: 5463 [1977]). See Table 4 below and FIG. 7 herein.

TABLE 4 C_(H)3 of anti-CD3 C_(H)3 of CD4-IgG Most Preferred MutantsT366Y Y407T T366W Y407A F405A T394W Y407T T366Y T366Y:F405A T394W:Y407TT366W:F405W T394S:Y407A F405W:Y407A T366W:T394S Preferred Mutants F405WT394S

Residue T366 is within hydrogen-bonding distance of residue Y407 on thepartner C_(H)3 domain. Indeed the principal intermolecular contact toresidue T366 is to residue Y407 and vice versa. Oneprotuberance-into-cavity pair was created by inverting these residueswith the reciprocal mutations of T366Y in one C_(H)3 domain and Y407T inthe partner domain thus maintaining the volume of side chains at theinterface (FIG. 9). Mutations are denoted by the wild-type residuefollowed by the position using the Kabat numbering system (Kabat et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md., ed. 5, [1991]) and then the replacement residuein single-letter code. Multiple mutations are denoted by listingcomponent single mutations separated by a colon.

Phagemids encoding anti-CD3 light (L) and heavy (H) chain variants(Shalaby et al., J. Exp. Med. 175: 217 [1992] and Rodrigues et al., Int.J. Cancer (Suppl.) 7: 45 [1992]) were co-transfected into humanembryonic kidney cells, 293S, together with a CD4-IgG variant encodingphagemid (Byrn et al., Nature 344: 667 [1990]) as previously described(Chamow et al., J. Immunol. 153: 4268 [1994]). The procedure isillustrated in FIG. 8 herein. The total amount of transfected phagemidDNAs was fixed whereas the ratio of different DNAs was varied tomaximize the yield of Ab/Ia chimera. The ratio (by mass) of Ia:H chain:Lchain input DNAs (15 ÿg total) was varied as follows: 8:1:3; 7:1:3;6:1:3; 5:1:3; 4:1:3; 3:1:3; 1:0:0; 0:1:3.

The products were affinity purified using Staphylococcal protein A(ProSep A, BioProcessing Ltd, UK) prior to analysis by SDS-PAGE followedby scanning LASER densitometry (FIGS. 10A-10E). Excess L over H chainDNA was used to avoid the L chain from being limiting. The identity ofproducts was verified by electroblotting on to PVDF membrane(Matsudaira, J. Biol. Chem. 262: 10035 [1987]) followed by aminoterminal sequencing.

Co-transfection of phagemids for L chain together with those for H chainand Ia incorporating wild-type C_(H)3 resulted in a mixture of Ab/Iachimera, IgG and Ia homodimer products as expected (Chamow et al., J.Immunol. 153: 4268 [1994]). See FIG. 10A. The larger the fraction ofinput DNA encoding antibody H plus L chains or Ia the higher thefraction of corresponding homodimers recovered. An input DNA ratio of6:1:3 of Ia:H:L yielded 54.5% Ab/Ia chimera with similar fractions of Iahomodimer (22.5%) and IgG (23.0%). These ratios are in good agreementwith those expected from equimolar expression of each chain followed byrandom assortment of H chains with no bias being introduced by themethod of analysis: 50% Ab/Ia chimera, 25% Ia homodimer and 25% IgG.

In contrast to chains containing wild-type C_(H)3, Ab/Ia chimera wasrecovered in yields of up to 92% from cotransfections in which theanti-CD3 H chain and CD4-IgG Ia contained the Y407T cavity and T366Yprotuberance mutations, respectively (FIG. 10B). Similar yields of Ab/Iachimera were obtained if these reciprocal mutations were installed withthe protuberance on the H chain and the cavity in the Ia (FIG. 10C). Inboth cases monomer was observed for the chain containing theprotuberance but not the cavity. Without being limited to any onetheory, it is believed that the T366Y protuberance is more disruptive tohomodimer formation than the Y407T cavity. The fraction of Ab/Ia hybridwas not significantly changed by increasing the size of bothprotuberance and cavity (Ab T366W, Ia Y407A). A second protuberance andcavity pair (Ab F405A, Ia T394W) yielded up to 71% Ab/Ia chimera using asmall fraction of Ia input DNA to offset the unanticipated proclivity ofthe Ia T394W protuberance variant to homodimerize (FIG. 10D). Combiningthe two independent protuberance-into-cavity mutant pairs (AbT366Y:F405A, Ia T394W:Y407T) did not improve the yield of Ab/Ia hybridover the Ab T366Y, Ia Y407T pair (compare FIGS. 10C and 10E).

The fraction of Ab/Ia chimera obtained with T366Y and Y407T mutant pairwas virtually independent of the ratio of input DNAs over the rangetested. Furthermore the contaminating species were readily removed fromthe Ab/Ia chimera by ion exchange chromatography (0-300 mM NaCl in 20 mMTris-HCl, pH8.0) on a mono S HR 5/5 column (Pharmacia, Piscataway,N.J.). This augurs well for the preparation of larger quantities Ab/Iachimeras using stable cell lines where the relative expression levels ofAb and Ia are less readily manipulated than in the transient expressionsystem.

The protuberance-into-cavity mutations identified are anticipated toincrease the potential applications of Fc-containing BsAb by reducingthe complexity of the mixture of products obtained from a possible tenmajor species (Suresh et al., Methods Enzymol. 121: 210 [1990]) down tofour or less. It is expected that the T366Y and Y407T mutant pair willbe useful for generating heteromultimers of other human IgG isotypessince T366 and Y407 are fully conserved and other residues at the C_(H)3domain interface of IgG₁ are highly conserved (see FIG. 6 herein).

The invention claimed is:
 1. A method of preparing a heteromultimercomprising a first polypeptide and a second polypeptide which meet at aninterface, wherein the interface of the first polypeptide comprises aprotuberance which is positionable in a cavity in the interface of thesecond polypeptide, the method comprising the steps of: (a) culturing ahost cell comprising nucleic acid encoding the first polypeptide andsecond polypeptide, wherein the nucleic acid encoding the firstpolypeptide has been altered from the original nucleic acid to encodethe protuberance or the nucleic acid encoding the second polypeptide hasbeen altered from the original nucleic acid to encode the cavity, orboth, wherein the culturing is such that the nucleic acid is expressed,and wherein the host cell is a yeast cell; and (b) recovering theheteromultimer from the host cell culture.
 2. The method of claim 1wherein the nucleic acid encoding the first polypeptide has been alteredfrom the original nucleic acid to encode the protuberance and thenucleic acid encoding the second polypeptide has been altered from theoriginal nucleic acid to encode the cavity.
 3. The method of claim 1wherein step (a) is preceded by a step wherein nucleic acid encoding anoriginal amino acid residue from the interface of the first polypeptideis replaced with nucleic acid encoding an import amino acid residuehaving a larger side chain volume than the original amino acid residueto form the protuberance.
 4. The method of claim 3 wherein the importamino acid residue is arginine (R), phenylalanine (F), tyrosine (Y) ortryptophan (W).
 5. The method of claim 1 wherein step (a) is preceded bya step wherein nucleic acid encoding an original amino acid residue inthe interface of the second polypeptide is replaced with nucleic acidencoding an import amino acid residue having a smaller side chain volumethan the original amino acid residue to form the cavity.
 6. The methodof claim 5 wherein the import amino acid residue is not cysteine (C). 7.The method of claim 5 wherein the import amino acid residue is alanine(A), serine (S), threonine (T), or valine (V).
 8. The method of claim 1wherein the first and second polypeptide each comprise an antibodyconstant domain.
 9. The method of claim 8 wherein the antibody constantdomain is a C_(H)3 domain.
 10. The method of claim 9 wherein theantibody constant domain is from an IgG.
 11. The method of claim 10wherein the IgG is human IgG₁.
 12. The method of claim 1 wherein theheteromultimer is a bispecific antibody.
 13. The method of claim 1wherein the heteromultimer is a bispecific immunoadhesin.
 14. The methodof claim 1 wherein the heteromultimer is an antibody-immunoadhesinchimera.
 15. The method of claim 3 wherein one original amino acidresidue from the first polypeptide has been replaced with an importamino acid residue.
 16. The method of claim 5 wherein one original aminoacid residue from the second polypeptide has been replaced with animport amino acid residue.
 17. The method of claim 1 wherein step (a) ispreceded by a step wherein the nucleic acid encoding the first andsecond polypeptide is introduced into the host cell.
 18. Aheteromultimer prepared by the method of claim
 1. 19. A compositioncomprising the heteromultimer of claim 18 and a pharmaceuticalacceptable carrier.
 20. A host cell comprising nucleic acid encoding theheteromultimer of claim 18, wherein the host cell is a yeast cell. 21.The host cell of claim 20 wherein the nucleic acid encoding the firstpolypeptide and the nucleic acid encoding the second polypeptide arepresent in a single vector.
 22. The host cell of claim 20 wherein thenucleic acid encoding the first polypeptide and the nucleic acidencoding the second polypeptide are present in separate vectors.
 23. Amethod of making a heteromultimer comprising culturing the host cell ofclaim 20 so that the nucleic acid is expressed and recovering theheteromultimer from the cell culture.
 24. The method of claim 23,wherein the heteromultimer is recovered from the cell culture media. 25.A method of preparing a heteromultimer comprising: (a) altering a firstnucleic acid encoding a first polypeptide so that an amino acid residuein the interface of the first polypeptide is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance on the first polypeptide; (b) altering a second nucleicacid encoding a second polypeptide so that an amino acid residue in theinterface of the second polypeptide is replaced with an amino acidresidue having a smaller side chain volume, thereby generating a cavityin the second polypeptide, wherein the protuberance is positionable inthe cavity; (c) introducing into a yeast host cell the first and secondnucleic acids and culturing the yeast cell so that expression of thefirst and second nucleic acid occurs; and (d) recovering theheteromultimer formed from the yeast cell culture.
 26. The method ofclaim 25 wherein the first and second polypeptide each comprise anantibody constant domain.
 27. The method of claim 25 wherein theantibody constant domain is a C_(H)3 domain.
 28. The method of claim 27wherein the antibody constant domain is from a human IgG.